Combinatorial DNA Libraries Research Articles

Protein design-scapes generated by microfluidic DNA assembly elucidate domain coupling in the bacterial histidine kinase CpxA

The randomization and screening of combinatorial DNA libraries is a powerful technique for understanding sequence-function relationships and optimizing biosynthetic pathways. Although it can be difficult to predict a priori which sequence combinations encode functional units, it is often possible to omit undesired combinations that inflate library size and screening effort. However, defined library generation is difficult when a complex scan through sequence space is needed. To overcome this challenge, we designed a hybrid valve- and droplet-based microfluidic system that deterministically assembles DNA parts in picoliter droplets, reducing reagent consumption and bias. Using this system, we built a combinatorial library encoding an engineered histidine kinase (HK) based on bacterial CpxA. Our library encodes designed transmembrane (TM) domains that modulate the activity of the cytoplasmic domain of CpxA and variants of the structurally distant "S helix" located near the catalytic domain. We find that the S helix sets a basal activity further modulated by the TM domain. Surprisingly, we also find that a given TM motif can elicit opposing effects on the catalytic activity of different S-helix variants. We conclude that the intervening HAMP domain passively transmits signals and shapes the signaling response depending on subtle changes in neighboring domains. This flexibility engenders a richness in functional outputs as HKs vary in response to changing evolutionary pressures.

Quality control and statistical evaluation of combinatorial DNA libraries using nanopore sequencing.

Protein engineering and synthetic biology applications increasingly rely on the assembly of modular libraries composed of thousands of different combinations of DNA building blocks. At present, the validation of such libraries is performed by Sanger sequencing analysis on a small subset of clones on an ad hoc basis. Here, we implement a systematic procedure for the comprehensive evaluation of combinatorial libraries, immediately after their creation in vitro, using long reads sequencing technology. After an initial step of nanopore sequencing, we use straightforward bioinformatics tools to tabulate the composition and synteny of the building blocks in each read. We subsequently use exploratory statistics to assess the library and validate its diversity before carrying downstream cloning and screening assays.

The Use of Thermal Dissociation for Selection of DNA Aptamers

A method of thermal dissociation of DNA–protein conjugates was developed for the selection of aptamers to human alpha-fetoprotein. The method is based on the identification of the DNA oligonucleotides from the combinatorial DNA libraries with the highest specificity to targets, which remained within the conjugate after two stages: a) washing of unbound sequences; and b) removal of the sequences bound to the DNA but displaying a low affinity to it. The undestroyed most stable complexes were subjected to the thermal dissociation with the involvement of the corresponding oligonucleotides in the next rounds of selection. A comparison of the proposed method with the standard washing demonstrated a more intensive enrichment of the library. Due to the tighter conditions for washing of the target-bound DNA oligonucleotides the use of thermal dissociation can increase the efficiency of selection of highly specific aptamers to their molecular targets.

Enzymatic Preparation of Modified DNA: Study of the Kinetics by Real-Time PCR

The effects of modified deoxyuridine triphosphates (mod-dUTPs) with different substituents at the C5 position of the pyrimidine cycle on the kinetics of PCR with Taq and Vent (exo-) DNA polymerases are studied. Substituents in mod-dUTP include carboxamide group and groups that are part of the side chains of alanine, valine, leucine, phenylalanine, tryptophan, or tyrosine. For each mod-dUTP, the yields of the target product are measured with the full substitution of dTTP. A fragment of bacterial DNA with a certain nucleotide sequence and a synthetic combinatorial DNA library of random nucleotide sequences are used as templates for amplification. For each mod-dUTP-template-polymerase combination, the correlation between the amplification efficiencies and yields of the target product are investigated. PCR product accumulation curves are influenced by both the template used and the presence of a modified substrate. The catalytic activity of Taq polymerase is higher when mod-dUTPs with short aliphatic substituents are used and decreases when the derivatives with long aliphatic, phenyl, and indole substituents are utilized. Vent (exo-) polymerase is less sensitive to the chemical structure of mod-dUTP. The dynamic measuring of DNA accumulation may be useful for optimizing the temperature-time PCR profiles individually for each of the mod-dUTP. The derivatives may be used in combination with Vent (exo-) polymerase to obtain modified DNA sequences for the method of selection of modified aptamers (mod-SELEX).

Preparation of Modified Combinatorial DNA Libraries via Emulsion PCR with Subsequent Strand Separation

Preparation of Modified Combinatorial DNA Libraries via Emulsion PCR with Subsequent Strand Separation

Preparation of Modified Combinatorial DNA Libraries via Emulsion PCR with Subsequent Strand Separation

A modification of the enzymatic method for the preparation of combinatorial random DNA libraries, which combines amplification in isolated microvolumes with the simultaneous incorporation of modified nucleotides and subsequent separation of DNA strands, was developed. Deoxyuridine triphosphate with hydrophobic substituents such as structural analogues of amino acid side chains in the C5 position of the pyrimidine ring was used to introduce modifications into DNA. To prevent competitive amplification, which reduces the representativeness of combinatorial libraries, PCR in inverse emulsion was used. The separation of the strands of PCR products was carried out. There were six single-stranded DNA libraries with complete substitution of deoxythymidine via modified analogues with various functional groups. These DNA libraries are suitable for generating aptamers to protein targets through additional hydrophobic interactions from the introductions of appropriate modifications, and are completely compatible with the SELEX aptamer selection methodology.

On the overlap assembly of strings and languages

This paper investigates properties of the binary string and language operation overlap assembly which was defined by Csuhaj-Varju, Petre and Vaszil as a formal model of the linear self-assembly of DNA strands: The overlap assembly of two strings, xy and yz, which share an "overlap" y, results in the string xyz. The study of overlap assembly as a formal language operation is part of ongoing efforts to provide a formal framework and rigorous treatment of DNA-based information and DNA-based computation. Other studies along these lines include theoretical explorations of splicing systems, insertion/deletion systems, substitution, hairpin extension, hairpin reduction, superposition, overlapping catenation, conditional concatenation, contextual intra- and intermolecular recombinations, template guided recombination, as well as directed extension by PCR. In this context, we investigate overlap assembly and its properties: closure properties of basic language families under this operation, decision problems, as well as the possible use of iterated overlap assembly to generate combinatorial DNA libraries.

Computational methods for the construction, editing, and error correction of DNA molecules and their libraries.

The field of synthetic biology is fueled by steady advances in our ability to produce designer genetic material on demand. This relatively new technological capability stems from advancements in DNA construction biochemistry as well as supporting computational technologies such as tools for specifying large DNA libraries, as well as planning and optimizing their actual physical construction. In particular, the design, planning, and construction of user specified, combinatorial DNA libraries are of increasing interest. Here we present some of the computational tools we have built over the past decade to support the multidisciplinary task of constructing DNA molecules and their libraries. These technologies encompass computational methods for [1] planning and optimizing the construction of DNA molecules and libraries, [2] the utilization of existing natural or synthetic fragments, [3] identification of shared fragments, [4] planning primers and overlaps, [5] minimizing the number of assembly steps required, and (6) correcting erroneous constructs. Other computational technologies that are important in the overall process of DNA construction, such as [1] computational tools for efficient specification and intuitive visualization of large DNA libraries (which aid in debugging library design pre-construction) and [2] automated liquid handling robotic programming [Linshiz et al., Mol Syst Biol 4:191, 2008; Shabi et al., Syst Synth Biol 4:227-236, 2010], which aid in the construction process itself, have been omitted due to length limitations.

Assembly of Designed Oligonucleotides: a useful tool in synthetic biology for creating high-quality combinatorial DNA libraries.

The method dubbed Assembly of Designed Oligonucleotides (ADO) is a powerful tool in synthetic biology to create combinatorial DNA libraries for gene, protein, metabolic, and genome engineering. In directed evolution of proteins, ADO benefits from using reduced amino acid alphabets for saturation mutagenesis and/or DNA shuffling, but all 20 canonical amino acids can be also used as building blocks. ADO is performed in a two-step reaction. The first involves a primer-free, polymerase cycling assembly or overlap extension PCR step using carefully designed overlapping oligonucleotides. The second step is a PCR amplification using the outer primers, resulting in a high-quality and bias-free double-stranded DNA library that can be assembled with other gene fragments and/or cloned into a suitable plasmid subsequently. The protocol can be performed in a few hours. In theory, neither the length of the DNA library nor the number of DNA changes has any limits. Furthermore, with the costs of synthetic DNA dropping every year, after an initial investment is made in the oligonucleotides, these can be exchanged for alternative ones with different sequences at any point in the process, fully exploiting the potential of creating highly diverse combinatorial libraries. In the example chosen here, we show the construction of a high-quality combinatorial ADO library targeting sixteen different codons simultaneously with nonredundant degenerate codons encoding various reduced alphabets of four amino acids along the heme region of the monooxygenase P450-BM3.

Functional Inhibition of Sugar Transport by a Designed Novel Protein

Designed ankyrin repeat proteins have been shown to be useful for different applications such as structural, mechanistic, clinical studies, as well as drug development. We have constructed a combinatorial DNA library encoding ankyrin repeat proteins by PCR-based assembly. The ankyrin proteins have both N- and C-terminal caps and five internal repeats (called N5C). The estimated diversity of the library is higher than 1020. Applying circular dichroism spectroscopy, four purified proteins exhibit high melting temperature (> 95 °C). We have tested the constructed library for the selection of specific ankyrin proteins targeting the melibiose transport in Escherichia coli. The melibiose permease is one of few overexpressed proteins during bacterial infection for gaining energy. Using cells with plasmid-encoded constructed library and chromosomally-encoded MelA, the alpha-galactosidase, and MelB, the melibiose permease, we have collected a group of N5C candidates that alter the melibiose fermentation with no inhibition of glucose fermentation, indicating no effect on the downstream utilization pathways. Three candidates show significant inhibition of melibiose transport. Further tests, using sugar transport assay on different genetic backgrounds, MelA activity assay, and protein-protein interaction assay, have been applied to identify specific effect. These preliminary studies suggested that one N5C protein possibly inhibits mel operon. These bio-reagents could be potentially applied to study the sugar transport during bacterial infection and also facilitate transporter proteins crystallography.

Label-free detection of gliadin food allergen mediated by real-time apta-PCR

Celiac disease is an immune-mediated enteropathy triggered by the ingestion of gluten. The only effective treatment consists in a lifelong gluten-free diet, requiring the food industry to tightly control the gluten contents of their products. To date, several gluten quantification approaches using antibodies are available and recommended by the legal authorities, such as Codex Alimentarius. However, whilst these antibody-based tests exhibit high sensitivity and specificity, the production of antibodies inherently requires the killing of host animals and is time-consuming and relatively expensive. Aptamers are structured single-stranded nucleic acid ligands that bind with high affinity and specificity to their cognate target, and aiming for a cost-effective viable alternative to the use of antibodies. Herein, we report the systematic evolution of ligands by exponential enrichment (SELEX)-based selection of a DNA aptamer against gliadin from a combinatorial DNA library and its application in a novel detection assay. Taking into account the hydrophobic nature of the gliadin target, a microtitre plate format was exploited for SELEX, where the target was immobilised via hydrophobic interactions, thus exposing aptatopes accessible for interaction with the DNA library. Evolution was followed using surface plasmon resonance, and following eight rounds of SELEX, the enriched DNA pool was cloned, sequenced and a clear consensus motif was identified. An apta-PCR assay was developed where competition for the aptamer takes place between the surface-immobilised gliadin and gliadin in the target sample, akin to an ELISA competitive format where the more target present in the sample, the less aptamer will bind to the immobilised gliadin. Following competition, any aptamer bound to the immobilised gliadin was heat-eluted and quantitatively amplified using real-time PCR, achieving a detection limit of approx. 2nM (100ngmL(-1)). The specificity of the selected aptamer was demonstrated and no cross-reactivity was observed with streptavidin, bovine serum albumin or anti-gliadin IgG.

J5 DNA Assembly Design Automation Software

Recent advances in Synthetic Biology have yielded standardized and automatable DNA assembly protocols that enable a broad range of biotechnological research and development. Unfortunately, the experimental design required for modern scar-less multipart DNA assembly methods is frequently laborious, time-consuming, and error-prone. Here, we report the development and deployment of a web-based software tool, j5, which automates the design of scar-less multipart DNA assembly protocols including SLIC, Gibson, CPEC, and Golden Gate. The key innovations of the j5 design process include cost optimization, leveraging DNA synthesis when cost-effective to do so, the enforcement of design specification rules, hierarchical assembly strategies to mitigate likely assembly errors, and the instruction of manual or automated construction of scar-less combinatorial DNA libraries. Using a GFP expression testbed, we demonstrate that j5 designs can be executed with the SLIC, Gibson, or CPEC assembly methods, used to build combinatorial libraries with the Golden Gate assembly method, and applied to the preparation of linear gene deletion cassettes for E. coli. The DNA assembly design algorithms reported here are generally applicable to broad classes of DNA construction methodologies and could be implemented to supplement other DNA assembly design tools. Taken together, these innovations save researchers time and effort, reduce the frequency of user design errors and off-target assembly products, decrease research costs, and enable scar-less multipart and combinatorial DNA construction at scales unfeasible without computer-aided design.

Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries

High-throughput genomics, proteomics and synthetic biology studies require ever more efficient and economical strategies to clone complex DNA libraries or variants of biological modules. In this paper, we provide a protocol for a sequence-independent approach for cloning complex individual or combinatorial DNA libraries, and routine or high-throughput cloning of single or multiple DNA fragments. The strategy, called circular polymerase extension cloning (CPEC), is based on polymerase overlap extension and is therefore free of restriction digestion, ligation or single-stranded homologous recombination. CPEC is highly efficient, accurate and user friendly. Once the inserts and the linear vector have been prepared, the CPEC reaction can be completed in 10 min to 3 h, depending on the complexity of the gene libraries.

A Spectrophotometric Microtiterplate Assay to Determine the Transphosphatidylation Potential of Phospholipase D

AbstractIn addition to the hydrolysis of the terminal phosphate ester bond in glycerophospholipids, phospholipases D (PLDs) are able to catalyze the exchange of the polar head group. The biocatalytic potential of PLDs strongly depends on the ratio of the transphosphatidylation to hydrolysis rate which, therefore, is an important criterion in the screening for efficient PLDs from natural sources or combinatorial DNA libraries. Here, we present a fast spectrophotometric assay that allows the determination of the rate of both hydrolysis and transphosphatidylation of PLD‐containing solutions including cell extracts in one microtiterplate. The assay is based on the reaction of phosphatidylcholine solubilized in Triton X‐100 micelles with ethanolamine and the determination of phosphatidic acid (PA) and choline. PA is determined via Fe(III) complexation and represents hydrolysis, while choline is determined by the conventional choline oxidase/peroxidase assay and yields the total conversion. The difference between both values corresponds to the transphosphatidylation product. The method is suitable for measuring reactions rates as well as product yields after defined time periods. As shown for E. coli cells expressing PLD from cabbage, the assay can be applied to extracts of cells grown and lysed in microtiterplates.

Differential Inhibitory Activities and Stabilisation of DNA Aptamers against the SARS Coronavirus Helicase

The helicase from severe acute respiratory syndrome coronavirus (SARS‐CoV) possesses NTPase, duplex RNA/DNA‐unwinding and RNA‐capping activities that are essential for viral replication and proliferation. Here, we have isolated DNA aptamers against the SARS‐CoV helicase from a combinatorial DNA library. These aptamers show two distinct classes of secondary structure, G‐quadruplex and non‐G‐quadruplex, as shown by circular dichroism and gel electrophoresis. All of the aptamers that were selected stimulated ATPase activity of the SARS‐CoV helicase with low‐nanomolar apparent K m values. Intriguingly, only the non‐G‐quadruplex aptamers showed specific inhibition of helicase activities, whereas the G‐quadruplex aptamers did not inhibit helicase activities. The non‐G‐quadruplex aptamer with the strongest inhibitory potency was modified at the 3′‐end with biotin or inverted thymidine, and the modification increased its stability in serum, particularly for the inverted thymidine modification. Structural diversity in selection coupled to post‐selection stabilisation has provided new insights into the aptamers that were selected for a helicase target. These aptamers are being further developed to inhibit SARS‐CoV replication.

Automating the DNA computer: solving n-Variable 3-SAT problems

In the decade since the first molecular computation was performed, it has been shown that DNA molecules can perform sophisticated, massively parallel computations avoiding the Von Neumann bottleneck. However, progress in the field has been slow. The largest problem solved to date is an instance of the 20-variable 3-CNF SAT problem. Performing the computation took more than two man-weeks to complete because every aspect of the computation was performed by hand. Molecular computations are extremely labor intensive and error prone---automation is necessary for further progress. The next step, (the second generation DNA computer--that of taking the laborious, laboratory bench protocols performed by hand, and automating them), has been achieved with the construction of an automated DNA computer dubbed EDNAC. It employs the same paradigm that was used to solve the labor-intensive instance of the 20-variable 3-CNF SAT problem. Using a combinatorial DNA library and complementary probes, EDNAC solves instances of the n-variable 3-CNF SAT problem. A 10 variable instance of the 3-CNF SAT problem was essayed. The computation took 28 h to perform. EDNAC correctly computed nine of the 10 variables, with a tenth variable remaining ambiguous. This result is comparable to current results in the molecular computation community. This research tested the critical properties, such as complexity, robustness, reliability, and repeatability necessary for the successful automation of a molecular computer.

Characterization of long RNA-cleaving deoxyribozymes with short catalytic cores: the effect of excess sequence elements on the outcome of in vitro selection

We previously conducted an in vitro selection experiment for RNA-cleaving deoxyribozymes, using a combinatorial DNA library containing 80 random nucleotides. Ultimately, 110 different sequence classes were isolated, but the vast majority contained a short14–15 nt catalytic DNA motif commonly known as 8–17. Herein, we report extensive truncation experiments conducted on multiple sequence classes to confirm the suspected catalytic role played by 8–17 and to determine the effect of excess sequence elements on the activity of this motif and the outcome of selection. Although we observed beneficial, detrimental and neutral consequences for activity, the magnitude of the effect rarely exceeded 2-fold. These deoxyribozymes appear to have survived increasing selection pressure despite the presence of additional sequence elements, rather than because of them. A new deoxyribozyme with comparable activity, called G15–30, was ∼2.5-fold larger and experienced a ∼4-fold greater inhibitory effect from excess sequence elements than the average 8–17 motif. Our results suggest that 8–17 may be less susceptible to the potential inhibitory effects of excess arbitrary sequence than larger motifs, which represents a previously unappreciated selective advantage that may contribute to its widespread recurrence.

Autoimmunity and tumor immunity induced by immune responses to mutations in self

Little is known about the consequences of immune recognition of mutated gene products, despite their potential relevance to autoimmunity and tumor immunity. To identify mutations that induce immunity, here we have developed a systematic approach in which combinatorial DNA libraries encoding large numbers of random mutations in two syngeneic tyrosinase-related proteins are used to immunize black mice. We show that the libraries of mutated DNA induce autoimmune hypopigmentation and tumor immunity through cross-recognition of nonmutated gene products. Truncations are present in all immunogenic clones and are sufficient to elicit immunity to self, triggering recognition of normally silent epitopes. Immunity is further enhanced by specific amino acid substitutions that promote T helper cell responses. Thus, presentation of a vast repertoire of antigen variants to the immune system can enhance the generation of adaptive immune responses to self.

Sequence-Independent Helical Wrapping of Single-Walled Carbon Nanotubes by Long Genomic DNA

Because of their nanometer sizes and molecular recognition capabilities, biological systems have garnered much attention as vehicles for the directed assembly of nanoscale materials.(1-6) One of the greatest challenges of this research has been to successfully interface biological systems with electronic materials, such as semiconductors and metals. As a means to address some of these issues, Sarikaya, Belcher, and others have used a combinatorial technique called phage display(7-9) to discover new families of peptides that showed binding affinities to various substrates. More recently, Zheng and co-workers used combinatorial DNA libraries to isolate short DNA oligomers (30-90 bases) that could disperse single-walled carbon nanotubes (SWCNT) in water.(10) Through a systematic analysis, they found that short oligonucleotides having repeating sequences of gunanines and thymines (dGdT)(n) could wrap in a helical manner around a CNT with periodic pitch.(11) Although helix formation around SWCNTs having regular pitches is an effective method for dispersing and separating CNTs, the need for specific repeating sequences limits use to non-natural DNA that must be synthesized with optimal lengths of less than 150 bases. In contrast, we demonstrate here that long genomic single-stranded DNA (>>100 bases) of a completely random sequence of bases can be used to disperse CNTs efficiently through the single-stranded DNA's (ssDNA) ability to form tight helices around the CNTs with distinct periodic pitches. Although this process occurs irrespective of the DNA sequence, we show that this process is highly dependent on the removal of complementary strands. We also demonstrate that although the helix pitch-to-pitch distances remain constant down the length of a single CNT, the distances are variable from one DNA-CNT to another. Finally, we report initial work that shows that methods developed to align long dsDNA can be applied in a similar fashion to produce highly dense arrays of aligned ssDNA-CNT hybrids.

Non-Shine-Dalgarno Initiators of Translation Selected from Combinatorial DNA Libraries

In a search for non-Shine-Dalgarno (non-SD) translational initiators, two combinatorial expression libraries (denoted R₁ and R₂) were constructed containing randomized decanucleotide regions placed at either 6 (R₁) or 11 (R₂) nucleotides upstream of a modified chloramphenicol acetyltransferase (CAT) gene. To prevent sporadic formation of SD-like sequences the content of G in the randomized region was restricted to 3% only. The two libraries were transformed in Escherichia coli cells and screened for chloramphenicol (Cm) resistance. More than 50 clones capable of tolerating Cm concentrations from 50 µg/ml to more than 800 µg/ml were selected. With few exceptions only, the non-SD sequences found in the Cm-resistant clones did not show any significant homology with other known non-SD initiators or enhancers of translation. Statistical (χ²) analysis of the distribution of nucleotides in the new non-SD translational initiators showed a different pattern from that of the conventional SD sequences. In few of the clones the yield of CAT exceeded that of the referent (SD-containing) construct. The most productive clones carried the decanucleotides ATTTACCTCC, CCAATCTAC, TTCAATATTT, and TATTCCCCCA, and the corresponding yield of CAT obtained with them was 2.70, 2.06, 2.12 and 1.32 times, respectively, higher than that of the SD-bearing construct.